Chuang D T, Ku L S, Kerr D S, Cox R P
Am J Hum Genet. 1982 May;34(3):416-24.
To detect heterozygotes for maple-syrup-urine disease (MSUD), activities of branched-chain-alpha-ketoacid (BCKA) dehydrogenase and its components in skin fibroblasts of two obligatory heterozygotes and amnion cells of a fetus at risk were measured. Intact heterozygous cells were found to decarboxylate [1-14C] alpha-ketoisovalerate at rates equal to or only slightly lower than normal subjects. The inability to differentiate heterozygotes from normals with the intact cell assay confirms earlier studies with intact leukocytes using [1-14C]leucine as substrate. By contrast, measurements of BCKA dehydrogenase activity with disrupted cell suspensions showed MSUD heterozygotes with 30%--60% of normal activity. Moreover, biphasic kinetics in heterozygous cells were observed with increasing substrate concentrations. The altered biphasic kinetics probably reflect expression of the normal allele in the early hyperbolic portion of the curve of the mutant allele in the later secondary rise at high substrate concentrations. Assays of component activities showed concordant E1 decarboxylase deficiency in both heterozygous- and homozygous-affected cells, whereas the E3, dihydrolipoyl dehydrogenase-component, activity was normal. The above results taken together appear to provide an approach to detection of the heterozygote in MSUD.
为检测枫糖尿症（MSUD）的杂合子，我们测定了两名 obligatory 杂合子皮肤成纤维细胞以及一名有患病风险胎儿的羊膜细胞中支链α-酮酸（BCKA）脱氢酶及其组分的活性。完整的杂合细胞以等于或仅略低于正常受试者的速率脱羧[1-14C]α-酮异戊酸。完整细胞检测无法区分杂合子与正常个体，这证实了早期使用[1-14C]亮氨酸作为底物对完整白细胞进行的研究结果。相比之下，对破碎细胞悬液中 BCKA 脱氢酶活性的测量显示，MSUD 杂合子的活性为正常活性的 30% - 60%。此外，在杂合细胞中观察到随着底物浓度增加出现双相动力学。这种改变的双相动力学可能反映了正常等位基因在曲线早期双曲线部分的表达以及突变等位基因在高底物浓度后期二次上升中的表达。组分活性测定显示，杂合子和纯合受影响细胞中 E1 脱羧酶均缺乏，而 E3（二氢硫辛酰胺脱氢酶组分）活性正常。综合上述结果，似乎为检测 MSUD 杂合子提供了一种方法。
